Modification of protein thiol is one of the most important post-translational modifications and it occurs depending on the redox state in cells. Protein S-nitrosylation is NO (nitric oxide)-dependent modification of protein thiols and is crucial for regulation of cellular functions such as transcription, protein expression, and signal transduction. -SulfoBiotics- Protein S-Nitrosylation Monitoring Kit allows to detect S-nitrosylated proteins by gel-electrophoretic analysis. This kit contains chemical reagents for blocking of free thiols on proteins, reducing of S-nitrosylated thiols, and labeling of the reduced thiols. After blocking free thiols of protein, S-nitrosylated thiols are selectively reduced by the reducing agent, and labeled with Protein-SHifter Plus, which is a novel maleimidyl compound consisted of a high molecular weight. When one molecule of Protein-SHifter Plus binds to a thiol group of protein, a mobility shift corresponding to about 15 kDa of molecular mass is observed by the gel-electrophoretic analysis. Thus, the number of S-nitrosylated thiol group on a protein can be clearly identified by SDS-PAGE through the mobility shift assay. In addition, the Protein-SHifter Plus moiety can be cleaved from the labeled protein in a gel with UV irradiation after gel-electrophoresis because Protein-SHifter Plus has a UV photocleavable moiety in the molecule. Therefore, the protein treated with UV irradiation can be transferred from the gel to PVDF membrane and detected on the membrane similar to the unlabeled protein by a specific antibody.
Figure 1 Schematic Protocol of Protein S-Nitrosylation Monitoring Kit
1. S. Hara, Y. Tatenaka, Y. Ohuchi and T. Hisabori, “Direct determination of the redox status of cysteine residues in proteins in vivo”, Biochem. Biophys. Res. Commun., 2015, 456(1) 339.
2. X. Wang, N. Kettenhofen, S. Shiva, N. Hogg, and M. Gladwin, “Copper dependence of the biotin switch assay : modified assay for measuring cellular and blood nitrosated proteins”, Free Radic. Biol. Med., 2008, 44, 1362.
3. M. T. Forrester, M. W. Foster, M. Benhar, and J. S. Stamler, “Detection of Protein S-Nitrosylation with the Biotin Switch Technique”, Free Radic. Biol. Med., 2009, 46(2), 119.
4. M. D. Kornberg, N. Sen, M. R. Hara, K. R. Juluri, J. V. K. Nguyen, A. M. Snowman, L. Law, L. D. Hester, and S. H. Snyder, “GAPDH Mediates Nitrosylation of Nuclear Proteins”, Nat. Cell Biol., 2010, 12(11), 1094.
5. Wang X, Shults NV, Suzuki YJ, “Oxidative profiling of the failing right heart in rats with pulmonary hypertension.”, PLoS ONE 12(5): e0176887.
Analysis of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) S-Nitrosylation in HeLa cell
1.HeLa cells were seeded on a 24-wells plate at the concentration of 5 x 10 cells/well and cultured overnight at 37oCin a 5% CO2 incubator (culture media : MEM).
2.The cells were washed using HBSS (500 μl) twice, and two different concentrations of S-nitrosocysteine solutions (1 mmol/l and 100 μmol/l) in PBS (500 μl) was added to each well.
3.The cells were incubated at 37oC for 45 minutes.
4.After the cells were washed using HBSS (500 μl) twice, Blocking Solution (200 μl) was added to each well. Then, the cells were dissolved by pipetting.
5.The cell lysate was transferred to each tube, and incubated at 37oC for 10 minutes.
6.Cold acetone (1 ml) was added to each tube, and the supernatants were removed after centrifugation of the tubes at 12,000 x g for 3 minutes.
7.Step 6 was repeated.
8.Cold 70% EtOH solution (1 ml) was added, and the supernatants were removed after centrifugation of the tubes at 12,000 x g for 3 minutes.
9.Lysis Buffer (20 μl) was added, and the cell pellet was dissolved by vortex and sonication.
10.RA Solution (4 μl) was added to Protein-SHifter Plus and mixed by pipetting.
11.The solution (2 μl) of Step 9 and Reaction Buffer B (4 μl) were added to the tube of Step 10, and the solution was mixed by pipetting.
12.The tube of Step 11 was incubated at 37oC for 30 minutes.
13.Loading Buffer ([10 (w/v) % sodium dodecyl sulfate, 50 (v/v) % glycerol, 0.2 mol/l Tris-HCl (pH 6.8) , 0.05 (w/v) % bromophenol blue], 2 μl) was added to the tube of Step 12 and mixed by pipetting.
14.The solution of Step 13 was used for SDS-polyacrylamide gel (10-20%) electrophoresis.
15.The gel was exposed with UV rays(302 nm) using a transilluminator for 10 minutes.
16.The separated proteins in the gel were electrophoretically transferred onto a PVDF membrane.
17.The GAPDH on the membrane was detected with anti-GAPDH antibody, HRP labeled secondary antibody, and luminol substrate.
Figure 2 Analysis of GAPDH S-Nitrosylation in HeLa cells